Borgnetto M E, Zunino F, Tinelli S, Kas E, Capranico G
Division of Experimental Oncology B, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Cancer Res. 1996 Apr 15;56(8):1855-62.
DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity. Since chromatin structure and genome organization are expected to influence drug-enzyme interactions and repair of drug-induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and amonafide in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencing by primer extension. VM-26 stimulated cleavage in the satellite III DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis in cloned DNA fragments with Drosophila topoisomerase II showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats. While stimulating similar levels of DNA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed for cleavage extent and reversibility in specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug-stimulated DNA cleavage in the chromatin of living cells.
不同拓扑异构酶II抑制剂刺激产生的DNA切割在体外表现出特征性的序列特异性。由于染色质结构和基因组组织有望影响药物与酶的相互作用以及药物诱导的DNA损伤修复，我们通过Southern印迹法和引物延伸基因组测序，研究了替尼泊苷（VM-26）、4-脱甲氧基-3'-脱氨基-3'-羟基-4'-表阿霉素（dh-EPI，一种阿霉素衍生物）、4'-（9-吖啶基氨基）-甲磺酰间茴香胺和氨萘非特在果蝇细胞组蛋白基因位点和卫星III DNA中刺激产生的拓扑异构酶II DNA切割位点。VM-26刺激卫星III DNA产生切割，而其他所研究的药物则没有。所有这四种药物均刺激组蛋白基因簇产生切割，但它们产生了药物特异性的切割强度模式。对组蛋白H2A基因启动子中dh-EPI和VM-26的切割位点进行了测序，结果显示它们是不同的。用果蝇拓扑异构酶II对克隆的DNA片段进行DNA切割分析表明，这些药物在体内和体外刺激相同的位点。链切口在体内错开4个碱基，主要的dh-EPI和VM-26位点的碱基序列与已知的体外药物序列特异性完全一致。此外，卫星III中的DNA切割恢复速度比组蛋白重复序列中的更快。虽然在总体基因组DNA中刺激产生相似水平的DNA断裂，但dh-EPI和VM-26在特定染色质位点的切割程度和可逆性明显不同。结果表明，活细胞染色质中药物刺激的DNA切割在定位、程度和可逆性方面存在高度异质性。
